Abstract

This thesis is focused on the characterization of Gram negative endotoxin as they play a key role in bacterial pathogenesis. Gram negative endotoxins or lipopolysaccharides (LPS) are glycoconjugates molecules exposed toward the external environment as they are the major components of Gram negative external leaflet. They have a structural role since they contribute to the cellular rigidity increasing the strength of cell wall and mediate the contacts with the external environment. Moreover, LPS can modulate the host immune response as they are recognised by immune mechanisms of defence. Structural elucidation of these molecules is an essential pre requisite in the comprehension of chemical structures that are responsible for bacterial pathogenesis. Lipopolysaccharides are build up according to a common structural architecture. They are composed of a hydrophilic hetero-polysaccharide (formed by core oligosaccharide and O-specific polysaccharide or O-chain) covalently linked to a lipophilic domain termed lipid A, which is embedded in the outer leaflet and anchors these macromolecules to the membrane through electrostatic and hydrophobic interactions. These three major domains are genetically, biologically and chemically distinct.
The endotoxins analysed in this work are extracted from opportunistic Cystic Fibrosis pathogens. These bacteria are responsible for feared infections that reduce life expectancy in CF patients.
The first part of the project was centred on the study of LPS involvement in bacterial adaptation after lung transplantation to a healthy not CF tissue. LPS were extracted from the most representative and problematic group of CF pathogens, the Burkholderia cepacia complex (Bcc). Burkholderia multivorans (genomovar II) and Burkholderia vietnamiensis (genomovar V) were respectively isolated from CF patients that underwent lung transplantation. For each clinical isolated, two paired clonal strains were recovered: one pre and the other post transplantation. Thus, in order to investigate the possible structural changes that possible occurred during the adaptation, for the first time LPSs from each strain, isolated in two different moments of the infection, were extracted and characterized. B. multivorans LPS isolated pre and post transplantation lacking the polysaccharidic moiety of the O-chain, revealed to be a lipooligosaccharide (LOS). The differences between the LOSs isolated from the two clonal strains were mainly found in the lipid A moiety, even though in the post transplantation strain there was an additional residue of Ara4N on the core portion. These chemical differences were responsible for the different biological activities found for LOSs. Generally, it was found a loss of inflammatory activity after lung transplantation when the two purified LOSs were tested as elicitors of TNF-alpha induction in human myelomonocytic U937 cells and of NF-KB induction in transfected TLR 4/MD2/CD 14 HEK cell lines. Even B. vietnamiensis, producing in both pre- and post transplantation strains LOSs, showed substantial differences in the lipid A moieties that justified an increased inflammatory activity for post transplantation strain when it was tested for TNF-alpha and NF-KB inductions. The features found in both studies revealed that lipid A moiety changes during bacterial adaptation. These data are not surprising since this LPS portion accounts for endotoxin virulence as it is recognized by the innate immune system of the host.
Further, from a post transplanted allograft, B. cenocepacia (genomovar III) the most virulent of Bcc members, was also collected, and its endotoxin characterized. This structural investigation constituted a complementary work to the previous study on B. cenocepacia ET-12 endotoxin already performed in this laboratory. Thus, it gives a clear aspect of the endotoxin phenotype produced by the most pathogenic strain of Bcc, when it colonized a survived CF patient.
An additional structural investigation was also performed on the second most pathogenic Bcc member, namely B. multivorans. The endotoxin was recovered from the most virulent strain of genomovar II, the C1576, that was responsible of a fatal outbreak in Glasgow, when only few children survived to adulthood. This strain produces a smooth type LPS, and its lipid A and O-chain moieties were characterized. In this case, differently from the other Burkholderia lipid A analyzed, bacteria produced a less phosphorylated blend of lipid A species. Even though lipid A is the highest conserved portion of LPS, little changes, as the presence or absence of charged residues or the number and the distribution of fatty acids, may occur in response to bacterial exigencies of adaptation. The O-chain portion was instead constituted by two polysaccharides characterized by two different repeating units. Moreover, a conformational study was executed on these polysaccharides in order to investigate about their supra molecular arrangements.
A conformational study and a MD simulation was also performed on the oligosaccharidic region of the LOS molecule extracted from the clinical isolated strain of B. cenocepacia ET-12, previously characterized. This analysis was performed with AMBER package in a solvent explicit model, thus mimicking real conditions. The investigation on three dimensional structure adopted by the most exposed moiety of B. cenocepacia endotoxin better clarified its spatial orientation. Moreover, data obtained give a real idea of the residues that are exposed toward the external environment, thus playing a key role in molecular recognition. The results carried out also gave information about the relative orientation of sugar rings and the conformational spaces energetically permitted for glycosidic bond rotations. These data can also contribute to justify and explain LPS role in increasing membrane rigidity and cell-wall strength.
The last part of this project was focused on the investigation of whether and, possibly, how P. aeruginosa genetic adaptation resulted in a bacterial strategy to evade the host immune sensing. With this aim, the impact of lipopolysaccharide, purified by P. aeruginosa strains isolated from acute and chronic infection of a CF patient, was analysed. Typically, P. aeruginosa infections are followed by a chronic persistence of the disease that cause permanent respiratory symptoms and decline in lung functions. The long term colonization of CF airways selects pathoadaptive variants with several features which differentiate late P. aeruginosa isolates from the initially acquired strain. Even though P. aeruginosa strains that initiates infections are characterized by a large arsenal of virulence factors, CF chronic infection are characterized by less virulent but more persistent phenotypes. LPS modification appears to be one of the main factors in the adaptation of this pathogen during chronic infections but no conclusive information were present so far on the putative lipid A changes in the acute to chronic evolution of the infection. The results obtained emphasize the reduced immunopotential of LPS extracted from late colonizer P. aeruginosa strains, demonstrating the lost of large arsenal of virulence factors during chronic infection.